Carboxypeptidase A catalyzes the enolization of the ketonic substrate analog (-)-2-benzyl-3-p-methozybenzoylpropionic acid stereospecifically. Sterochemical analysis of the exchange process shows that the substrate carbonyl oxygen is bound to the active site metal ion, and the active site Glu-270 residue acts as a protein abstracting group. We plan to study the enolization reaction using various metallocarboxypeptidases in which the active site zinc ion has been replaced by Co(II), Ni(II) ad Mn(II), allowing us to examine the catalytic properties of the enzyme-bound nucleophilic group and the active site metal ion without the complication of the formation and breakdown of a multiplicity of reaction intermediates. Additionally, we will detemine whether derivatives of the ketone substrate bearing good leaving groups at the 2-position of the propionic acid moiety can function either as substrates for carboxypeptidase A or, alternatively, whether they might function as "suicide" inhibitors, trapping the active site nucleophile with concomitant inhibition of enzymatic activity. New stereospecific syntheses of both enantiomers of 2-mercapto-3-phenylpropionic acid will allow us to probe te structural requirements for the competitive inhibition of carboxypeptidase activity. Finally, while an acyl-enzyme intermediate in the reaction of carmobxypeptidase A with the ester O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate at sub-zero temperature in aqueous-organic solvents has been detected, it has not been feasible to monitor the kinetics of both catalytic steps, acylation and deacylation, over a wide pH range. O-(trans-Dimethylaminocinnamoyl)-L-beta-phenyllactate appears to be a promising substrate for such studies. We hope to probe the chemistry of the various metallocarboxypeptidases with this or a related substrate to learn more about the role of the metal ion in both acylation and deacylation. The approaches used with the A enzyme will be extended to the examination of carboxypeptidase B. The results obtained should provide an intriguing comparison of two structurally related but different metal-containing exopeptidases.